Project Summary/Abstract The epidermal growth factor receptor (EGFR) has key regulatory roles in epithelial wound healing, cell growth and cell survival in many mammalian cell types. EGFR is hijacked by many human viruses, including human papillomaviruses (HPVs), to promote their infectious entry into host cells. Yet, specific mechanisms by which EGFR activation leads to virion internalization are poorly understood. HPV types 16 and 31 interact with EGFR and the EGFR-interacting partner, the annexin A2 tetramer (A2t), at the HK plasma membrane. Our scientific premise is based on findings from our lab and from other groups: functional inhibition of EGFR or A2t proteins significantly reduces HPV entry and infection in HKs. Our CENTRAL HYPOTHESIS is that EGFR signaling promotes the translocation of A2t to the external cell membrane to initiate the infectious entry of HPV into host cells. We will define the role that EGFR and its signaling plays in promoting HPV uptake in HKs, and determine whether EGFR is directly involved in onco-HPV internalization. We will also examine how A2t cooperates with EGFR signaling to facilitate infectious entry of onco-HPV. Epithelial wounding plays an important, but poorly understood, role in papillomavirus infections in vivo. Notably, EGFR and A2t are activated in epithelial wound settings. We will use infectious entry assays (detection of viral transcription or pseudo-genome expression) coupled with biochemical and genetic cell manipulations to define the mechanisms of HPV infection of normal host HKs. Quantitative multi-parameter confocal, super-resolution and video microscopy will permit us to visualize the fate of HPVs and localize the viruses with cell proteins under genetic and biochemical conditions that prevent infectious entry. We will combine the techniques described above with cell culture and mouse models of infection where epithelial wounding can be induced to examine how wounding contributes to HPV infections. Three inter-related SPECIFIC AIMS will address our central hypothesis. In Aim 1 we will determine function of EGFR in onco-HPV entry into HKs. In Aim 2 we will define how the interplay between EGFR and A2t promotes the uptake of onco-HPVs into HKs. In Aim 3 we will assess the roles of EGFR and A2t in HPV entry in keratinocytes using in vitro and in vivo wound models. Hypotheses regarding specific aspects of HPV-HK interactions, EGFR and AnxA2 activities and HPV entry are posed. Whether our theories are supported or refuted, we will increase our understanding of the molecular mechanisms of HPV-host cell interactions, the biology of HK uptake, and EGFR regulation in HKs, each of which are poorly understood. This fundamental science program may also inform strategies for preventing persistent onco-HPV infections, a major risk for morbidity and malignant progression. As EGFR and AnxA2 are important for other virus infections, our results may be applicable to these pathogens as well. Lastly, we will gain needed insight into the cooperative functions of EGFR and A2t, both of which are dysregulated in many human diseases, including squamous cell cancers. !